Fluorescence is a primary means by which biomolecules are tracked and quantitated. Fluorescent tags are used in DNA sequencing, gene expression analysis using microarrays, flow cytometry and its variants, RT-PCR, and a host of other applications. One application of special interest, and increasing importance, is intracellular imaging. Bimolecular binding/debinding events have been monitored by fluorescence resonance energy transfer (FRET), whereby event-driven changes in the distance between a fluorophore and quencher lead to changes in fluorescence intensity. Covalent attachment of fluorophores to surface antibody markers on white blood cells is the basis of cytometry; likewise covalent attachment of fluorescent tags has been used to visualize every organelle within a cell, and virtually every process that cells undergo. Despite their numerous strengths, organic fluorophores have several limitations. (i) They are highly susceptible to photobleaching and photodecomposition as excited states are both better oxidants and better reductants than the ground state. (ii) The fluorescence emission envelope is broad, limiting the number of spectrally orthogonal tags. (iii) The fluorescence is typically excited in the visible, a region where biological samples exhibit intrinsic background fluorescence. (iv) different colors of fluorophores often have vastly different structures and chemical properties, necessitating different attachment and handling protocols.
The development of fluorescent nanoparticulate semiconductors (quantum dots) has extended the utility of fluorescence-based optical detection tags. (Chan, et al., Science 1998, 281, 2016-18; Bruchez, et al., Science 1998, 281, 2013-16.) The disclosure of Chan, et al., and all other patents, patent applications, and publications referred to herein are incorporated by reference herein in their entirety. Quantum dots exhibit far less photobleaching than organic fluorophores, and in the visible, have narrower emission bandwidths. A consequence of the narrower emission envelope, though, is the narrower excitation envelope in the visible; as a result, ultraviolet light is required to excite multiple tags, which is less than optimal for biological systems. Moreover, bandwidths of quantum dots increase considerably as the particles emit in the red and especially in the near-IR, and accordingly, many fewer colors are available. Nevertheless, quantum dots have been used extensively for intracellular imaging applications, with promising results. Recent reports have used quantum dots to track the binding and endocytosis of epidermal-growth factor and for long-term imaging of quantum dots that were endocytosed or attached to biotinylated surface proteins of living cells. However, the bulk of the literature describes non-targeted approaches to cellular delivery. A peptide translocation domain was used to introduce various ratios of 5 colors of quantum dots into cell subsets to produce 10 unique codes. Various reports describe tracking of cells injected into mice after being encoded by quantum dots, including using a lipofection reagent and transduction peptides. Development of Xenopus embryos was also tracked after injection of micelle-encapsulated quantum dots. Unfortunately, endocytosed quantum dots are sequestered and not able to participate in further intracellular labeling and quantum dots delivered by transfection and electroporation are prone to aggregation. Microinjection allows delivery of unaggregated quantum dots, but is a serial process requiring much skill. Additionally, the long-term imaging with UV light can cause degradation of the quantum dots, resulting in spectral shifts and cytotoxicity. Likewise, the introduction of metal nanoparticles into cells has been described. Metal nanoparticles were shown to be successfully introduced inside living cells in 1990, when electron microscopy was used to examine nuclear uptake of colloidal gold microinjected into the cytoplasm. (Feldherr, et al, J Cell Biol 1990, 111, 1-8; Feldherr, et al., J Cell Biol 1991, 115, 933-39.) The art was advanced by using video-enhanced color microscopy to track the nuclear targeting ability of peptide modified colloidal gold. Several reports have taken advantage of the strong plasmon resonance from Ag and Au nanoparticles by modifying them with biological molecules to track the dynamics of membrane-transporter protein on living cells in real time. The absence of photobleaching in both techniques allows long-term imaging without any degradation of the particles. In theory, particle size, shape and composition may be controlled to allow multiplexed plasmon resonance imaging experiments, but in practice, the width of features coupled with difficulty in making all particles a given size reduce the number of colors to 2-3.
Raman scattering is readily excited using monochromatic far-red or near-IR light, photon energies too low to excite the inherent background fluorescence in biological samples. Since Raman spectra typically cover vibrational energies from 200-3500 cm−1, and since individual vibrations typically have very narrow bandwidths, i.e. <50 cm−1, one could envisage measuring a dozen (or more) reporters simultaneously, all with a single light source; however, normal Raman is very weak, limiting its utility for use in bioanalytical chemistry. In SERS, molecules in very close proximity to nanoscale roughness features on noble metal surfaces (gold, silver, copper) give rise to million- to trillion-fold increases [known as enhancement factor (EF)] in scattering efficiency. SERS can also be used to detect molecules adsorbed to individual metal nanoparticles and has been used to demonstrate the detection of a single molecule.